PROJECT SUMMARY The epithelial stem cell of the stomach is poorly characterized, which is surprising given its role in many gastric diseases. High doses of the Selective Estrogen Receptor Modulator (SERM) tamoxifen (HD-Tam) kill parietal cells and causes a proliferative, metaplastic gastric response which follows the pattern seen in patients chronically infected with Helicobacter pylori, but in a more synchronous and controllable model, with the effect peaking at 3 days post injections. Published studies using HD-Tam show that this response is dependent on a ?-ERK?CD44??-STAT3 signaling axis and combines proliferation both from expansion of the isthmal stem cell (iSC) and contribution from mature zymogenic cells which reprogram to re-enter the cell cycle, termed recruited stem cells (rSCs). While these proliferating populations are inseparable in their response to any metaplasia-causing agent, we recently discovered that highly specific diphtheria toxin (DT)-mediated parietal cell death induces iSC expansion but no metaplasia or rSC generation. These two models now allow us to specifically characterize iSCs and rSCs to determine their relative mechanisms of proliferation. Our Aims are: 1) Characterize mechanisms of iSC expansion/recovery and determine the specific role of CD44 in iSC expansion both in vivo and in human and murine gastric organoids (?gastroids?). We will also sort pure iSCs following DT and mixed iSCs+rSCs following HD-Tam and run RNA-Seq to characterize each population. 2) Examine new metabolic pathways governing each proliferative population using rapamycin and metformin, both of which are seen to decrease proliferation following HD-Tam. These drugs will be tested in vivo following HD-Tam and DT as well as in gastroids, along with a drug screen to identify other proteins involved in their signaling pathways. 3) We are now able to determine whether metaplastic rSC generation increases risk of gastric tumorigenesis over increased iSC proliferation alone. We will follow an established protocol using the carcinogen MNU to drive tumorigenesis in mice while treating with cycles of DT or HD-Tam while monitoring the entire course of tumorigenesis non-invasively with Near Infrared Fluorescence live imaging with novel probes found to track gastric cancer in vivo. We will track specific mutations and proteins necessary for this cancer progression and focus on any candidates found with our large biobank of human gastric cancer tissues. Our DT and HD-Tam models allow us to separate iSC and rSC proliferation in the stomach for the first time, finally allowing for detailed characterization of each population. Our experiments aim to use this opportunity to attribute our known proliferative axis more specifically to iSC and/or rSC expansion, define new metabolic pathways involved in governing proliferation in each population, and run unbiased screens to identify additional modulators of these pathways. Finally, our models allow us to test a new theory for how solid tissue cancers arise, investigating whether rounds of reprogramming and cell cycle re-entry of mature, post-mitotic cells (rSC generation) increase risk for tumorigenesis over increased iSC proliferation alone.